Fluorescence polarization immunoassay for amphetamine/methamphetamine

ABSTRACT

This disclosure relates to a method and reagents for determining amphetamine and methamphetamine in a biological fluid such as urine. In particular, this disclosure relates to a fluorescence polarization immunoassay procedure for determining the presence of amphetamine and methamphetamine in a single assay and to a novel class of tracer compounds employed as reagents in such procedures. The procedure described includes pretreatment of the biological sample to eliminate cross-reactants such as β-hydroxyphenethylamine by preincubating the sample solely with an aqueous periodate solution having a pH from about 4.0 to about 7.5 without adjustment to an alkaline pH.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates generally to fluorescence polarizationimmunoassays and reagents useful therein, and particularly to such anassay for amphetamine and methamphetamine. The invention provides apreincubation step which is effective to eliminate cross reactivity toβ-hydroxyamines.

2. Background Art

Amphetamine and methamphetamine are sympathomimetic phenethylaminederivatives having central nervous system stimulant activity. Thesedrugs have been used for the treatment of obesity, narcolepsy, andhypotension. Because of their stimulant effects, the drugs are commonlysold illicitly and abused. Physiological symptoms often associated withvery high amounts of ingested amphetamine and methamphetamine includeelevated blood pressure, dilated pupils, hypethermia, convulsions, andacute amphetamine psychosis.

The biological fluid tested most frequently is urine. Other biologicalfluids have not been extensively investigated for use in assays for thedetection of amphetamine and methamphetamine. In the past, amphetamineshave been detected by a number of techniques, including thin-layerchromatography (TLC), gas chromatography (GC), and high performanceliquid chromatography (HPLC). These methods generally involve chemicalextractions of the drugs, complicated procedures requiring highlytrained personnel and lengthy assay times.

In general, competitive binding immunoassays have provided a preferablealternative to chemical methods such as GC and HPLC. Typically,competitive binding immunoassays are used for measuring ligands in atest sample. Generally, a "ligand" is a substance of biological interestto be determined quantitatively by a competitive binding immunoassaytechnique. The ligands compete with a labeled reagent, or "ligandanalog," or "tracer," for a limited number of receptor binding sites onantibodies specific to the ligand and ligand analog. The concentrationof ligand in the sample determines the amount of ligand analog whichbinds to the antibody: the amount of ligand analog that will bind isinversely proportional to the concentration of ligand in the sample,because the ligand and the ligand analog each bind to the antibody inproportion to their respective concentrations.

An accurate and reliable immunoassay requires that antibody"cross-reactivity" (recognition of compounds other than the desiredligand or ligands) be minimized. In the case of assays for amphetamineand methamphetamine it is known that derivatives of β-phenethylamine,such as β-hydroxyphenethylamine compounds, may be strong interferants.One such β-hydroxyphenethylamine, the drug phenylpropanolamine, is foundfrequently in decongestants sold over the counter. U.S. Pat. No.3,856,469 discloses removal of β-hydroxyphenethylamine interference froma sample intended for amphetamine or methamohetamine analysis bytreating the sample at a pH greater than 8.0 with an amount of aqueousperiodate in the presence of ammonium hydroxide. In addition torequiring sample treatment at a basic pH, the aqueous pretreatment inU.S. Pat. No. 3,856,469 is suggested as useful only preceeding sampleevaluation by thin layer chromatography and immunoassays byradioimmunoassay, electron spin resonance technique or enzyme technique.

Fluorescence polarization provides an alternative quantitative orqualitative means for measuring the amount of tracer-antibody conjugateproduced in a competitive binding immunoassay. Fluorescence polarizationtechniques are based on the principle that a fluorescent labeledcompound, when excited by plane polarized light, will emit fluorescencehaving a degree of polarization inversely related to its rate ofrotation. Accordingly, when a tracer-antibody conjugate having afluorescent label is excited with plane polarized light, the emittedlight remains highly polarized because the fluorophore is constrainedfrom rotating between the time that light is absorbed and emitted. Incontrast, when an unbound tracer is excited by plane-polarized light,its rotation is much faster than the corresponding tracer-antibodyconjugate and an excited population of molecules is randomized much morequickly. As a result, the light emitted from the unbound tracermolecules is depolarized.

To date no fluorescence polarization assay for determining amphetamineand/or methamphetamine in a single assay has been disclosed.

Accordingly, a need exists for providing a method and reagents forperforming a reliable and accurate fluorescence polarization assay forboth amphetamine and methamphetamine in biological fluids such as urine.A further need exists for conducting aqueous periodate pretreatment ofurine samples to be tested for amphetamine and/or methamphetaminewithout the addition of pH raising constituents, such as bases.

SUMMARY OF THE INVENTION

The present invention relates to a method for determining amphetamineand methamphetamine utilizing fluorescence polarization techniques. Inparticular the method of the present invention involves preincubation ofa urine sample to be tested for amphetamine and/or methamphetaminewithout adjustment of the sample's pH to alkaline conditions.Particularly, a sample is treated solely with an aqueous periodatesolution, having a pH from about 4.0 to 7.5, to eliminate undesirablecompounds which cross-react with antibodies specific for amphetamine andmethamphetamine and the ligand analogs thereof.

The treated sample is intermixed with a composition comprising firstfluorescein or fluorescein derivative tracer compound coupled to aligand analog of amphetamine, a second fluorescein or fluoresceinderivative tracer compound coupled to a ligand analog of methamphetamineand a first antibody capable of specifically recognizing and bindingamphetamine and the first tracer compound and a second antibody capableof specifically recognizing and binding methamphetamine and the secondtracer compound. The amount of the first and second tracer compoundsbound to the first and second antibodies, respectively, is determined byfluorescence polarization techniques as a measure of the amount ofamphetamine and methamphetamine in the sample. The first tracer compoundis preferably of the formula: ##STR1## and the second tracer compound ispreferably of the formula ##STR2## wherein Q is fluorescein or afluorescein derivative, preferably a carboxyfluorescein or4'-aminomethylfluorescein, T is a linking group selected from SO₂, HN,HN(CH₃)₃ O, COCH₂, CO(CH₂)₂ CONH, HN(CH₂)₂ or HN(CH₂)₂ NHCOCH₂.

The antibodies to amphetamine and methamphetamine employed in the assayare raised in response to amphetamine and methamphetamine derivativesattached to a protein carrier, preferably bovine serum albumin.

The present invention further relates to a stabilized reagent kit usefulfor determining amphetamine and methamphetamine in a single assayincluding a novel fluorescence reagent having tracers of both formulas(I) and (II) and salts thereof, which are useful as reagents in theabove-described method. Other components of the reagent kit inaccordance with the invention are an aqueous pretreatment solutionhaving an amount of periodate effective in eliminating undesirablecross-reactivity to β-hydroxyphenethylamines and an antibody reagentwith a composition comprising a first antibody capable of specificallyrecognizing and binding amphetamine and a second antibody specificallyrecognizing and binding methamphetamine. In the case of automatedfluorescence polarization assays utilizing automated dispensing meanssuch as a pipette, the present invention provides for a washing of thedispensing means with an aqueous periodate solution to minimize samplecarryover resulting from urine adhesion to the dispensing means. Thepreferred aqueous wash solution is from about 0.1 to 0.25 molar sodiumperiodate.

Further objects and attendant advantages of the invention will be bestunderstood from a reading of the following detailed description takentogether with the drawings and the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following Figures the symbol "F1" represents a fluoresceinmoiety, and the various other symbols are noted in the DetailedDescription.

FIG. 1 shows the general structure of the class of phenethylamines to bequantitatively or qualitatively determined in accordance with thepresent invention.

FIGS. 2a and 2b show a general structural formula for the tracers andthe immunogens of the present invention. FIG. 2c shows a generalstuctural formula for the class of reactants used to prepare tracers andimmunogens in accordance with the present invention.

FIG. 3 shows the alternate structural formulae and names of thefluorescein moiety included in the tracers of the present invention.

FIG. 4 shows various linkages that couple the fluorescein moiety to theprecursor in FIG. 2, when FIG. 2 represents a precursor for the tracers.

FIGS. 5 and 6 show examples of structures of preferred tracers ofamphetamine and methamphetamine in accordance with the presentinvention.

FIGS. 7 and 8 show examples of structures of preferred immunogens usedto raise antibodied to amphetamine and methamphetamine in accordancewith the present invention.

FIGS. 9(a-f) shows exemplary structures of haptens useful to form inaccordance with the present invention.

cl DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "ligand", as used herein, refers to a molecule, to which abindingprotein, such as a receptor or an antibody, can be obtained orformed. The ligands of interest in the present invention arephenethylamines, more particularly, amphetamine and methamphetamine.Such ligands are protein-free compounds, generally of low molecularweight, which do not induce antibody formation when injected into ananimal but which are reactive to antibodies. Ligands which arechemically modified for conjugation to a carrier protein are termedhaptens. Antibodies to haptensare generally raised by first conjugatingthe haptens to a protein carrier and injecting the conjugate productinto an animal. The resulting antibodies may be isolated byconventional, well-known antibody isolation techniques.

The term "ligand-analog", as used herein, refers to a mono- orpolyvalent radical, a substantial portion of which has the same spatialand polar organization as the ligand to define one or more determinantor epitopic sites capable of competing with the ligand for the bindingsites of a receptor. A characteristic of such a ligand-analog is that itpossesses sufficient structural similarly to the ligand of interest asto be recognized by the antibody against the ligand. For the most part,the ligand-analog will have the same or substantially the same structureand charge distribution (spatial and polar organization) as theligand(s) of interest (for purposes of the present invention,amphetamine and methamphetamine) for a significant portion of themolecular surface. Frequently, the linking site for a hapten will be thesame in preparing the antigen for production of antibodies as that usedin the tracer for linking to the ligand, the same portion of the ligandanalog which provides the template for the anitbody will be exposed bythe ligand analog in the tracer.

The present invention involves the use of fluorescein and derivatives offluorescein. A necessary property of fluorescein and its derivatives forthe usefulness of the tracer compounds herein is the fluorescence.Fluorescein exists in either of two tautomeric forms, illustrated inFIG. 3, depending on the acid concentration (pH) of the environnment. Inthe open (acid) form, there are a number of conjugated double bondswhich makethat form of flourescein (and compounds containing afluorescein moiety) capable of absorbing blue light and emitting greenfluorescence after an excited state lifetime of about 4 nanoseconds.When the open and closed forms coexist, the relative concentration ofmolecules in the open and closed forms is easily altered by adjustmentof the pH level. Generally, the tracer compounds of the presentinvention exist in solution as salts such as sodium, potassium, ammoniumand the like, allowing the compounds to exist in the open, fluorescentform, when employed in the analytical methods of the present invention.The specific salt present will depend onthe buffer employed to adjustthe pH level. For example, in the presence ofa sodium phosphate buffer,the compounds of the present invention will generally exist in the openform, as a sodium salt.

As used herein, the term "fluorescein," either as an individual compoundoras a component of a larger compound, is meant to include both the openand closed forms, if they exist for a particular molecule, except in thecontext of fluorescence. An open form is necessary for the fluorescencetooccur.

The numbering of carbon atoms of the fluorescein molecule varies,dependingupon whether the open or closed form of the molecule isconsidered. Accordingly, the literature concerning fluorescein and itscompounds is not uniform as to carbon atom numbering. In the closedform, the carbon para to the carboxylic acid group on the phenyl ring isnumbered 5. For purposes of this disclosure, the numbering of the closedform (as illustrated in FIG. 3) is adopted because the raw materialsused in the syntheses are most popularly numbered with that system. Thecarbon atom offluorescein and its derivatives para to the carboxyl groupis therefore numbered "6" for the purposes of the present disclosure.

A tracer in solution which is not complexed to an antibody is free torotate in less than the time required for absorption and re-emission offluorescent light. As a result, the re-emitted light is relativelyrandomly oriented so that the fluorescence polarization of a tracer notcomplexed to an antibody is low, approaching zero. Upon complexing witha specific antibody, the tracer-antibody complex thus formed assumes therotation rate of the antibody molecule which is slower than that of therelatively small tracer molecule, thereby increasing the polarizationobserved. Therefore, when a ligand competes with the tracer for antibodysites, the observed polarization of fluorescence of the resultingmixture of the free tracer and tracer-antibody complex assumes a valueintermediate between that of the tracer and that of the tracer-antibodycomplex. If a sample contains a high concentration of the ligand, theobserved polarization value is closer to that of the free tracer, i.e.,low. If the test sample contains a low concentration of the ligand, thepolarization value is closer to that of the bound tracer, i.e., high. Bysequentially exciting the reaction mixture of an immunoassay withvertically and then horizontally polarized light and analyzing only thevertical component of the emitted light, the polarization offluorescence in the reaction mixture may be accurately determined. Theprecise relationship between polarization and concentration of theligand to be determined is established by measuring the polarizationvalues of calibrators with known concentrations. The concentration ofthe ligand canbe extrapolated from a standard curve prepared in thismanner.

The particular tracers formed in accordance with this invention havebeen found to produce suprisingly good assays, as will be demonstratedinfra.

The Reagents

The objective in designing a Fluorescence Polarization Immunoassay is tohave competition between the desired phenethylamines and the tracers forthe recognition sites of the antibody. Great variations in the structureof the haptens and tracers are allowed in achieving this goal. Forpurposes of this invention, "haptens" are precursors of the immunogensor tracers, comprising generally a substituted phenethylamine derivativeand a linking group to the protein carrier or fluorescein compound.

1. Pretreatment Reagent

An important aspect of the present invention is the elimination ofcross-reactivity to β-hydroxyphenethylamines in a fluorescencepolarization assay by pretreating the test sample with an effectiveamountof an aqueous periodate solution. Specifically, the aqueousperiodate solution causes cleavage of the side chain between the α andβ-carbon when there is a hydroxy(--OH) attached to the α-carbon. Thus,the compound no longer competes for the binding sites.

The pretreatment reagent in accordance with the reagent kit of thepresent invention includes an aqueous periodate solution having a pHfrom about 4 to 7.5. Preferably, the pretreatment solution includes 0.1to 0.25M of sodium periodate having a pH range from about 4.0 to 5.0.Most preferably the sodium periodate solution includes about 0.125Msodium periodate having a pH of about 4.5. Suprisingly, it has beenfound that pretreatmentof a test sample can be conducted without theneed for pH adjustment of thetest sample to alkaline conditions withcompounds such as hydroxides.

2. The Tracers

a. The Structure of the Tracers

Useable tracers can be produced from a wide variety of phenethylaminederivatives. The tracers of the present invention have the generalstructural formula shown in FIG. 2, where Q represents a fluoresceinmoiety or a fluorescein derivative.

The tracer is a phenylethylamine derivative that is linked to afluoresceinderivative by, for example, an amido, amidino triazinylamino,carbamido, thiocarbamido, carbamoyl, thiocarbamoyl, or sulfonylcarbamylgroup, as shown in FIG. 4. The tracers are prepared by linking theappropiate fluorescein derivative to a phenylethylamine derivativecontaining an amino, carboxylic acid, hydroxy, imidate, hydrazide,isocyanate, thioisocyanate, chloroformate, chlorothioformate,chlorosulfonyl, or the like group, as will be discussed in the contextof the synthetic method and the Examples below.

By way of example, any of the following fluorescein derivatives can beused:

    ______________________________________                                        FlNH.sub.2   fluorescein amine                                                FlCO.sub.2 H carboxyfluorescein                                               FlNHCOCH.sub.2 I                                                              iodoacetamidofluorescein                                                      FlCH.sub.2 NH.sub.2                                                                        aminomethylfluorescein                                                         ##STR3##                                                                     2,4-dichloro-1,3,5-triazin-2-yl                                               amino-fluorescein (DTAF)                                                       ##STR4##                                                                     4-chloro-6-methoxy-1,3,5-triazin-2-                                           ylamino fluorescein (Methoxy DTAF)                               FlNCS        fluorescein isothiocyanate                                       ______________________________________                                    

The preferred tracers in accordance with the present invention arerepresented by formulas I and II herein. Most preferably the tracershave the structural formula shown in FIGS. 5 and 6 in whichcarboxyfluorescein and 4'-aminomethylfluorescein are linked toamphetamine and methamphetamine ligand-analogs, respectively.

b. The Synthesis of the Tracers

The tracers of the present invention are made by coupling a fluoresceinmoiety, or a derivative of fluorescein, to the general structure shownin FIG. 2 when X is NH₂, COOH, CNOR, OH or SO₂ Cl.

The fluorescein moiety can be linked to the amino, carboxyl,chlorosulfonyl, imidate or alkoxy functional group by an amide, anamidine, an urea, a thiourea, a carbamate, a thiocarbamate,triazinylamino, sulfonamide, or sulfonylcarbamate linkage, as shown inFIG. 4. In the presently preferred embodiment for amphetamine, thefluorescein derivative is 5-carboxyfluorescein and this is coupled to aprecursor of the tracer shown in FIG. 2c. The 5-carboxyfluorescein iscoupled to 4-(3-aminopropoxy)-amphetamine (protected on thephenethylaminenitrogen by a t-butoxycarbonyl group) by first forming theactive ester of 5-carboxyfluorescein. The preferred active ester isN-hydroxysuccinimide active ester and the preferred method is viaN,N'-dicyclohexylcarbodiimideactivation in dry pyridine. Otheractivating groups, such as 1-hydroxybenzotriazole, p-nitrophenol, andimidazole, can be used; and other solvents, such as dimethylformamideand dimethylsulfoxide, can be used. The reactants are preferably coupledunder conditions for forming amide linkages, and it is most preferredthat active ester procedures be used. The N-BOC group is then removed bybrief exposure to an approximately 1:1 solution of CF₃ CO₂ H and CH₂CL₂.

The preferred embodiment for the methamphetamine tracer is shown in FIG.6,where the fluorescein derivative is 4'-aminomethylfluorecein. This iscoupled to a precursor shown in FIG. 2c.

The methamphetamine derivative is converted to its active ester. Thepreferred active ester is a derivative of2-ethyl-5-phenylisoxazolium-3'-sulfonate also known as Woodward'sReagent K. Other activating groups, such as 1-hydroxybenztriazole,p-nitro-phenol,carbonyldiimidazole and N-hydroxysuccinimide can be used;and other solvents, such as dimethylformamide and dimethylsulfoxide canbe used. Thepreferred solvent is a mixture of dimethylformamide andtriethylamine; other solvents such as pyridine and dimethylsulfoxide canalso be used. The reactants are preferably coupled under conditions forforming amide linkages, and it is most preferred that active esterprocedures be used. The N-BOC group is then removed by brief exposure toa 1:1 solution of CF₃ COOH and CH₂ Cl₂.

Usable tracers can be prepared from a variety of phenylethylaminederivatives.

All phenylethylamine derivatives that have a terminal amino group, suchas amino, hydrazinyl, hydrazido or the like, are coupled tocarboxyfluorescein by the active ester method or the mixed anhydridemethod, and coupled to fluorescein isothiocyanate, DTAF or alkoxy DTAFby simply mixing the two materials in solution. The amino group can beconverted to the isocyanate and thioisocyanate groups by reaction withphosgene and thiophosgene, respectively. These are then condensed withfluoresceinamine or 4'-aminomethylfluorescein to produce the tracer.

All phenylethylamine derivatives that have a terminal chlorosulfonylgroup are coupled to 4'-aminomethylfluorescein or fluoresceinamine bysimply mixing the two materials in solution and using a base to removethe acid that is generated.

All phenylethylamine derivatives that have a terminal carboxylic acidgroup, such as carboxylic acid, (aminohydroxy)alkylcarboxylic acid orthe like, are coupled to 4'-aminomethylfluorescein or aminofluoresceinby the active ester method.

c. Combination of Tracers

According to the present invention, the preferred tracer reagent is acomposition comprising salts of a first tracer and a second tracer.Generally, the first tracer is a salt of a ligand analog to amphetamineand the second tracer is a salt of a ligand analog to methamphetamine.Thecombination of individual tracers for amphetamine and formethamphetamine provides the advantage of detection of both drugs(amphetamine/methamphetamine) while maintaining high specificity, lowcross-reactivity, high sensitivity and accuracy. Numerous combinationsof amphetamine tracers with mehtamphetamine tracers formed in accordancewiththe above described procedures may be used. Preferably, the firstand second tracers are salts of sodium, potassium, ammonium and thelike. Mostpreferably, the first and second tracers exist in the reagentsolution as sodium salts and the first tracer is the ligand analog ofamphetamine shown in FIG. 5 and the second tracer is the ligand analogof methamphetamine shown in FIG. 6. The tracer formula presentlypreferred isabout 150 nanomolar of the mixed tracers in 0.1 molar sodiumphosphate buffer at pH 7.5; 0.1% sodium azide; and 0.01 bovine gammaglobulin.

3. The Antibodies

The antibodies of the present invention are prepared by developing aresponse in animals to the immunogens described below. The immunogen isadministered to animals such as rabbits or sheep by a series ofinjections, in a manner well-known to those skilled in the art.

a. The Structure of the Immunogens

Usable antibodies can be produced from a variety of phenethylaminederivatives. Immunogens prepared from phenethylamine compoundsfunctionalized at the para position shown in FIG. 2, can produceantibodies in animals. Such antibodies are useful in an assay forphenethylamines according to the invention when combined with theappropriate tracer.

The immunogens of the present invention have the general structuralformulashown in FIG. 2 and are prepared by coupling a phenethylaminecompound of the class shown in FIG. 2 with a protein or a proteinderivative, as will be discussed in the context of the synthetic methodand the Examples below. The structural formula shown in FIG. 2 ispreferred because the best recognition of the desired phenylethylaminecompounds occurs when thering is substituted at a position as distant aspossible from the sidechain(i.e., para position).

In a preferred form of the invention, the immunogen is prepared bycouplingthe aforedescribed substituted phenethylamine compound withbovine serum albumin. Various other protein carriers may also be used togood advantage, e.g., keyhole limpet hemocyanin, egg ovalbumin, bovinegamma-globulin, thryoxine-binding globulin, and so forth. Alternatively,synthetic poly(amino acids) having a sufficient number of availableamino groups can be employed, as can other synthetic or naturalpolymeric materials bearing functional groups reactive with amphetamineor methamphetamine haptens. The most preferred immunogens according tothe present invention are shown in FIGS. 7 and 8.

b. The Synthesis of the Immunogens

The immunogens of the present invention are made by coupling anamphetamineor methamphetamine derivative to a poly(amino acid) as seengenerally in FIG. 2.

In a preferred embodiment, the poly(amino acid) is bovine serum albumin(BSA), and the hapten can be selected from one of the exemplarystructuresis FIG. 9 (a-f). These reactants are preferably coupled underconditions normally used to form amide, sulfonamide, urea, andalkylamine linkages, and such conditions are well known to those skilledin the art. It is mostpreferred when carboxylic groups are employed as apartner in the coupling reaction that active ester procedures be used,as these are the most effective in forming the desired amide linkages inthis context.

Before coupling the hapten to the poly(amino acid), the amine on thesidechain is protected. The protecting groups, for example:trifluoroacetyl or BOC (t-butylcarbamate), are added under conditionsknown to one skilled in the art.

The immunogens are prepared by coupling a hapten, having itsphenethylaminogroup protected and bearing an --NH₂, --CO₂ H, --CONHNH₂,--CNOR, --CHO, --Br, --I, --NCO, --NCS, --OCOCl, SO₂ Cl or --OCSCl groupin the para position, to a poly(amino acid). The --NH₂ can be coupled byreacting the amine with succinic anhydride, activating the resultingcarboxyl group and adding this to the poly(amino acid), or by activatingthe carboxylic acid group on the polyamino acid in the presenceof the--NH₂ group. The activation of the carboxylic acid groups on thepoly(amino acid) can be accomplished by mixing the hapten and thepoly(amino acid) with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), N,N'-dicyclohexylcarbodiimide (DCC),1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methyl-p-toluenesulfonate, or the like. The --CO₂ H case is also coupled by theactivation method (EDC) or the active ester method, as described belowin the tracer synthesis section. The --Br and --I cases also producealkylated amines on the poly(amino acid) but by direct coupling of thealkyl-halide to the amine on the poly(amino acid). The sulfonylchloride, isocyanate (--NCO), isothiocyanate (--NSC), chloroformate(--OCOCl) and chlorothioformate (--OCSCl) cases produce sulfonamide,urea, thiourea, carbamate and thiocarbamate linkages, respectively. Thisis accomplished by direct coupling of the hapten to the poly(aminoacid).

After coupling the sidechain amine-protected hapten to the protein, theprotecting group is removed to provide the free amine or the amine in asalt form. When the protecting group employed is trifluoroacetyl, it canbe removed by treatment with aqueous base, by exposure to aqueous sodiumborohydride, or other conditions known to one skilled in the art. Whentheprotecting group is BOC (t-butylcarbamate) it can be removed byaqueous acids, non-aqueous acids, or other procedures known to oneskilled in the art.

The syntheses of the above haptens are accomplished in very similarways. [FIG. 2c shows an immunogen precursor class in accordance with apreferredembodiment of the method of the present invention.] The idealstarting material is a phenylethylamine, such as amphetamine ormethamphetamine. The sidechain amine functionality must be renderedunreactive by a protecting group if the X moiety is SO₂ Cl. If X isBrCH₂ CONH, ICH₂ CONH, ClCONH, OCNH, or H₂ N, then the protecting groupcould be introduced before or after the following nitration reaction.Nitration for the latter group of haptens is accomplished by exposure ofthe protected or unprotected phenylethylamine to cold fuming nitricacid. After catalytic reduction of the nitro group to an amimo group, itis thencondensed with succinic anhydride, bromoacetyl bromide, orphosgene (or a phosgene-equivalent). Carboxyl-containing haptens areactivated using methods described above. The bromoacetamido derivativesare coupled to protein in the presence of aqueous potassium or sodiumiodide. In the casewhere X is SO₂ Cl, protein coupling is effected byexposing an aqueousor aqueous-organic solution of protein to thechlorosulfonyl phenylethylamine derivative. After conjugation, theprotecting groups are removed by methods known to one skilled in theart, and the immunogens arepurified either by size exclusionchromatography or dialysis.

c. Combination of Antibodies

According to the present invention, the preferred antibody reagent is acomposition comprising a first antibody raised in response to animmunogendescribed above, capable of recognizing and binding toamphetamine, and a second antibody, capable of recognizing and bindingmethamphetamine. Numerous combinations of antibodies raised in responseto amphetamine or methamphetamine immunogens, in accordance with theabove-described procedures, can be used provided that the antibodies arespecific for amphetamine and/or methamphetamine. Most preferably, theantibody reagent includes an amount of the antibody raised in responseto the immunogen in FIG. 7 and an amount of the antibody raised inresponse to the immunogen in FIG. 8.

Rabbit, sheep or any other animal serum can serve as the source ofantibodies for the antibody agent. The preferred antisera formulacomprises rabbit serum diluted with 0.1 molar sodium phosphate buffer atpH 7.5; 0.1% sodium azide; 0.01% bovine gamma globulin; and 2% ethyleneglycol (volume/volume).

4. Wash Reagent

It has been surprisingly determined that providing a phenethylaminefluorescence assay reagent kit with an aqueous periodate wash reagentimproves assay reliability and accuracy. Specifically, it has been foundthat providing a wash solution with about 0.1 to 0.25M aqueous periodateeliminates urine adhesion to dispensing means such as a probe, pipette,orsyringe. It is to be understood that urine adhesion to the dispensingmeanscan result in sample contamination yielding false positive resultsfor samples tested subsequent to a phenethylamine-containing sample. Inthe case of highly automated assaying apparatus, such as the ABBOTT TDx®Clinical Analyzer, which test large numbers of samples sequentially,eliminating urine "carryover" between samples is highly desirable.Preferably, the reagent kit is provided with a wash solution includingabout 0.125M sodium periodate.

The Assay

The particular tracers and antibodies of the present invention have beenfound to produce excellent results in fluorescence polarization assaysforthe desired phenethylamine. FIG. 1 shows the general structure of theclassof phenethylamines that can be quantitatively and/or qualitativelydetermined in accordance with the present invention. For example, oneassay of the present invention provides a more rapid and accurateamphetamine/methamphetamine assay method than prior art methods becauseitrequires no specimen treatment before analysis and the assay systemhas minimal cross-reactivity to amphetamine-like compounds.

The amphetamine/methamphetamine assay, in accordance with the analyticalmethods of the preferred embodiment of the present invention, involvespretreating a urine sample containing or suspected of containingamphetamine and/or methamphetamine with an effective amount of anaqueous periodate solution having a pH from about 4 to 7.5 for a periodof time sufficient to eliminate undesired cross-reactivity. Preferably,the sampleis pretreated with 0.1 to 0.25 molar aqueous sodium periodatesolution for about 1 to 9 minutes, most preferably 4 to 5 minutes at atemperature range from about 31° to about 36° C.

The pretreated sample is then mixed with tracer and antibody reagentsspecific to amphetamine and to methamphetamine. Amphetamine ormethamphetamine and the tracers compete for limited antibody sites,resulting in the formation of antibody-ligand complexes. By maintaininga constant concentration of tracer and antibody, the ratio of antibodycomplex to tracer-antibody complex formed upon incubation is directlyproportional to the amount of amphetamine and/or methamphetamine in thesample. Therefore, upon exciting the mixture with plane polarized lightand measuring the polarization of the fluorescence emitted by a traceranda tracer-antibody complex, one is able quantitatively orqualitatively to determine the amount of amphetamine and/ormethamphetamine in the sample.

The results can be quantified in terms of net millipolarization units,span(in millipolarization units) and relative intensity. The measurementof millipolarization units indicates the maximum polarization when amaximum amount of the tracer is bound to the antibody in the absence ofamphetamine or methamphetamine. The amount of tracer bound to theantibodyis directly proportional to the net millipolarization. Forpurposes of the present invention, a net millipolarization value of over190 is ideal, buta value in the range of about 150 to about 220 isacceptable. The span is an indication of the difference between the netmillipolarization at the points of the maximum and the minimum amount oftracer bound to the antibody. A larger span provides for a betterquantitative analysis of data. For the purposes of this invention, aspan of at least about 60 millipolarization units is preferred. Theintensity is a measure of the amplitude of the fluorescence signal thatis above the background fluorescence. Thus, a higher intensity will givea more accurate measurement. The intensity is determined for thepreferred tracers of the invention as the sum of the verticallypolarized intensity plus twice the horizontally polarized intensity. Theintensity can range from a signal ofabout three times to about thirtytimes the background noise depending uponthe concentration of the tracerand other assay variables. For the purposesof the present invention, anintensity of at least eight to ten times that of noise background ispreferred.

Table I shows the results obtained with the preferred antibodies raisedin response to immunogens (FIGS. 7 and 8) and tracer compounds (FIGS. 5and 6) of the present invention in terms of span, millipolarizationunits and intensity. As seen from the data in Table I, an assay usingthe antibody produced from the immunogen of FIG. 7 in combination withthe tracer of FIG. 5 provides excellent results for an amphetamineassay. For assay of methamphetamine, a combination of antisera derivedfrom an immunogen of FIG. 8 with tracer of FIG. 6 provides excellentresults.

One aspect of the present assay that is unique is the combination ofantisera produced from immunogens 7 and 8 with tracers 5 and 6 toproduce an assay with a net polarization of 210 and a span over 70 foreither amphetamine or methamphetamine. This is the most preferredconfiguration of the assay.

                  TABLE l                                                         ______________________________________                                        Hapten Used In                                                                Immunogen For                                                                 Antibody   Tracer  Net Polarization                                                                           Span  Intensity                               ______________________________________                                        FIG. 7     FIG. 5  213.40       130.71                                                                              2365.4                                  FIG. 8     FIG. 6  190.51       126.85                                                                              2505.9                                  ______________________________________                                    

The pH at which the method of the present invention is conducted must besufficient to allow the fluorescein moiety of the tracers to exist intheir open form. The pH may range from about 3 to 12, more usually inthe range of from about 5 to 10, most preferably from about 6 to 8.Various buffers may be used to achieve and maintain the pH during theassay procedure. Representative buffers include borate, phosphate,carbonate, tris, barbital and the like. The particular buffer employedis not critical to the present invention, but phosphate buffer ispreferred. The cation portion of the buffer will generally determine thecation portion of the tracer salt in solution.

The preferred method of the improved assay of the present invention isdiscussed in detail in Example 5. The assay is a "homogenous assay,"whichmeans that the end polarization readings are taken from a solutionin whichbound tracer is not separated from unbound tracer. This is adistinct advantage over heterogeneous immunoassay procedures where thebound tracermust be separated from the unbound tracer.

As described previously herein, the reagents for the fluorescencepolarization assay of the present invention comprise antibodies specificfor amphetamine and methamphetamine, fluorescein tracer analogs ofamphetamine and methamphetamine and a periodate pretreatment solution.Additionally, conventional amphetamine/methamphetamine assay solution,including a dilution buffer, d,l amphetamine calibrators andd,l-amphetamine controls are preferably prepared.

The preferred procedure is especially designed to be used in conjunctionwith the Abbott TDx® Clinical Analyzer available from AbbottLaboratories, Irving, Tex. It is to be understood that when the AbbottTDx® Analyzer is used, the assay is fully automated from pretreatment tofinal reading. However, manual assay can be performed. In the case ofautomated and manual assays, the sample is mixed with the pretreatmentsolution in dilution buffer and a background reading is taken. Thetracer is then mixed with the assay. The antibody is then finally mixedinto the test solution. After incubation, a fluorescence polarizationreading is taken and processed. In the case of both manual and automatedassays, the present method eliminates the need for sample pH adjustment.

EXAMPLE 1 Preparation of Methamphetamine ImmunogenD,L-Methamphetamine-N-Trifluoroacetamide

460 mg each of D- and L-methamphetamine were combined in a 25 mLroundbottom flask which was then cooled in an ice-water bath. Over a 15minute period, 8 mL of trifluoroacetic anhydride was added gradually in2 mL-portions to the stirred solution of amines. After stirring thesolutionfor an additional 1 hour at 0° C., the cooling bath was removedand the pale yellow solution was stirred for 20 hours at roomtemperature. At the end of this period, the solution was poured overabout 50 mg of ice and the resulting two-phase solution was diluted with50 mL of diethylether into a separatory funnel. The aqueous layer wasremoved, and the organic phase was washed twice with 50 mL portions ofwater, once with50 mL saturated aqueous sodium acetate, and again with50 mL of water. After drying over sodium sulfate, evaporation of thediethyl ether gave 1.46 gm of a pale yellow oil. An analytical samplewas isolated by preparative thin layer chromatography (TLC). Thus, 310mg of the crude product was eluted with ethyl acetate on three 1mm-thick, 20×20 cm silica gel plates. The upper dark bands at R_(f) 0.78were collected to afford 244 mg of a pale yellow liquid.

4'-Chlorosulfonyl-D,L-Methamphetamine-N-Trifluoroacetamide

D,L-Methamphetamine-N-trifluoroacetamide (244 mg) was dissolved in 3 mLchloroform in a 25 mL roundbottom flask and then cooled in an ice-waterbath. Chlorosulfonic acid (3 mL.) was then added dropwise over 5 minutesto the stirred solution. After stirring for an additional 4 hours atthis temperature, the pale yellow solution was added dropwise to 50 gmice in aseparatory funnel. The cold aqueous solution was extracted twicewith 30 mLportions of chloroform. The organic extracts were combined,dried over sodium sulfate, and evaporated to afford 315 mg of a paleyellow oil.

Preparation of a 4'-Sulfonyl-D,L-Methamphetamine Immunogen

Bovine serum albumin, BSA, (134 mg.) was dissolved in 4.4 ml of 0.1Mdisodium phosphate (pH 8.0). After 5 minutes, 0.7 mL ofdimethylformamide (DMF) was added to the protein solution.4'-chlorosulfonyl-D,L-methamphetamine-N-trifluoroacetamide (52 mg) wasdissolved in 500 ul of DMF and added on one portion to the aqueoussolution of BSA. The initial turbidity disappeared after a brief periodofstirring at room temperature. Conjugation was allowed to continue fora total of 24 hours at this temperature. After transferring the solutionto a dialysis bag, the solution was dialyzed against 5 changes ofdeionized water (4 L each for 6 hours per change). Titration usingpicrylsulfonic acid revealed 31% hapten incorporation into the BSA. Thecontents of the dialysis bag were collected and diluted with 2 mL ofmethanol and 1.8 mL piperidine and allowed to stand for 18 hours at roomtemperature. The pH of this hydrolysis solution was approximately 12.Dialysis as described above was again performed, followed bylyophilization to give 91 mg of a fluffy white material.

EXAMPLE 2 Preparation of Amphetamine Immunogen4-Nitro-D,L-Amphetamine-HCl

Fuming nitric acid (30 mls) was cooled to -35° C. D,L-Amphetaminesulfate (5.0 g) was added slowly (25 min.) with stirring. The reactionmixture (red) was stirred at this temperature for 2 hours, then allowedtowarm to -15° C. (solution color changed from red to yellow), andstirred at -15° C. for 30 minutes. The solution was then poured into 125ml ice water and extracted with 125 ml of benzene. The aqueous extractwas made basic (pH=11) with 6M NaOH and extracted three times with150 mlportions of benzene. There organic extracts were combined, dried overmagnesium sulfate, and filtered. To the filtrate was added 250 ml ofmethanol, then HCl (gas) was bubbled into the stirred solution untilsolution pH was 3. Solvent was removed in vacuo and crude productrecrystallized from ethanol-diethyl ether to afford 3.05 g of paleyellow powder.

4'-Nitro-D,L-Amphetamine-N-Trifluoroacetamide

4'-Nitro-D,L-Amphetamine.HCl (3.0 g) was dissolved in pyridine (40 mls),cooled to 0° C. and trifluoroacetic anhydride (9.8 mls) added. Thesolution was stirred at 0° C. for 2 hours, then poured into 200 ml icewater. The resulting precipitate was isolated via filtration andresidual solvent removed in vacuo to afford 3.46 g of tan powder.

4'-Amino-D,L-Amphetamine-N-Trifluoroacetamide

4'-Nitro-D,L-Amphetamine-N-Trifluoroacetamide (600 mg) was dissolved in50 ml absolute ethanol, 5% palladium on carbon (300 mg) was added, andthe mixture hydrogenated under 21 psi of hydrogen in a Parr hydrogenatorat room temperature for 2 hours. The catalyst was removed by filtrationand solvent removed in vacuo to afford 421 mg of a pale yellow oil.

4'-Hemisuccinamido-D,L-Amphetamine-N-Trifluoroacetamide

4'-Amino-D,L-Amphetamine-N-Trifluoroacetamide (1.00 g) was dissolved in7.0ml chloroform, and succinic anhydride (614 mg) was added, followed bytriethylamine (0.623 ml). The reaction was stirred for 21/2 hours atroom temperature under a nitrogen atmosphere, then diluted with 75 ml ofwater,pH adjusted to 4 with 1M HCl, and extracted 3 times with 100 mlportions ofethyl acetate. The ethyl acetate fractions were combined,dried over magnesium sulfate, and solvent removed in vacuo to afford1.31 g of off-white solid.

4'-Succinamido-D,L-Amphetamine Immunogen

4'-Hemisuccinamido-D-L-Amphetamine-N-Trifluoroacetamide (50 mg) andN-Hydroxysuccinamide (20 mg) were dissolved in 0.500 ml of anhydrousdimethylformamide. Dicyclohexylcarbodiimide (36 mg) was added and thereaction stirred for about 2 hours and added dropwise to a solution ofbovine serum albumin (125 mg) dissolved in 3.15 ml of 0.1M sodiumphosphate (pH=7.5) and 1.35 ml of 1,4-dioxane. The resulting cloudysolution was stirred for 16 hours at room temperature, then transferredtodialysis tubing and dialyzed against 0.1M sodium phosphate (pH=7.5) (4liters) for 2 hours, then against deionized water (2 changes of 4 literseach). The mixture was then centrifuged and supernatant lyophilized toafford 105 mg of a fluffy white powder. A portion of the product (45 mg)was dissolved in a solution of piperadine (2.5 mls), methanol (5 mls),andwater (20 mls). After stirring for 45 minutes at room temperature,the solution was transferred to dialysis tubing, dialyzed againstdeionized H₂ O (3 changes of 2 liters each), and lyophilized to afford34 mg offluffly white solid.

EXAMPLE 3 Preparation of Amphetamine Tracer 4-hydroxyphenylacetone

Hydrobromic acid (48%, 250 mL) was heated to approximately 120° C. Overa period of two minutes, a 9.85 g sample of 4-methoxyphenylacetone wasadded dropwise. After 15 minutes, the reaction mixture was chilled to30° C. and diluted to 500 mL with distilled water. The resulting mixturewas extracted twice with 250 mL portions of diethyl ether. The combinedorganic extracts were washed with brine, dried over magnesium sulfate,and the solvent was removed in vacuo. The resulting oil was purifiedimmediately by column chromatography over silica gel, using 40% ethylacetate and 60% hexane. Appropriate fractions were pooled and solventsremoved in vacuo to yield a thick yellow oil.

4-(3-chloropropoxy)phenylacetone

A 4.50 g portion of p-hydroxyphenylacetone, prepared above, wasdissolved in 80 mL of anhydrous dimethylformamide under a dry nitrogenatmosphere. With stirring 1.26 g of sodium hydride (as a 60% dispersionin oil) was added to the solution. After 3 minutes, 9.40 g of1-chloro-3-iodopropane was quickly added. After stirring forapproximately 18 hours, the reactionwas diluted with 300 mL hexane and100 mL diethyl ether. The resulting mixture was washed with distilledwater, 5% sodium hydroxide solution, andbrine. The remaining organicphase was dried and evaporated to dryness in vacuo. The yellow oil waspurified by column chromatography over silica gel, using 22% ethylacetate and 78% hexane. Appropriate fractions were pooled and solventsremoved in vacuo to yield 3.06 g of a yellow oil.

4-(3-chloropropoxy)-1-(2-aminopropyl)benzene

A 1.36 g sample of 4-(3-chloropropoxy)phenylacetone, prepared above, wasdissolved in 200 mL of methanol. To this was added 7.71 g of ammoniumacetate followed by 0.76 g sodium cyanoborohydride with stirring. After5 hours, the solvent was evaporated in vacuo, the residue was dissolvedin 150 mL diethylether and 200 ml of 1N hydrochloric acid solution. Theaqueous phase was separated and washed with a second portion ofdiethylether, basified to a pH of 10-12 with 6N sodium hydroxide, andextracted with three, 100 mL portions of diethylether. The basic etherextracts were combined, dried, and evaporated to dryness in vacuo toyield1.11 g of a yellow oil.

4-(3-chloropropoxy)-1-(2-(N-t-butyloxycarbonyl)amino-propyl)-benzene

A 1.0 g sample of 4-(3-chloropropoxy)-1-(2-aminopropyl)benzene, preparedabove, was dissolved in 50 mL of dichloromethane. To this was added,1.92 g of di-t-butyl-dicarbonate. After stirring at room temperature for18 hours, the solvent was removed in vacuo and the residue was dissolvedin 35 ml diethylether and 15 mL of 5% sodium carbonate and then stirredfor 2hours. The organic phase was separated, washed with brine, driedover magnesium sulfate, filtered, and evaporated to dryness in vacuo.The resulting yellow oil was purified by column chromatography oversilica gelusing 20% ethyl acetate and 80% hexane. Appropriate fractionswere pooled and solvents were removed in vacuo to yield 1.74 g of awhite solid.

4-(3-Iodopropoxy)-1-(2-(N-t-butyloxycarbonyl)aminopropyl)benzene

A solution of 1.30 g of4-(3-chloropropoxy)-1-(2-(N-t-butyloxycarbonyl)aminopropyl)-benzene in40 mL anhydrous 2-butanone was prepared. To this was added 1.80 g sodiumiodide. The resulting mixture was refluxed for 24 hours, cooled to roomtemperature, and diluted with 200 mL diethylether. The mixture waswashed with 5% sodium thiosulfate solution, twice with brine, dried overmagnesium sulfate, filtered, and evaporated to dryness in vacuo to yield1.14 g of a white solid.

4-(3-Aminopropoxy)-1-(2-(N-t-butyloxycarbonyl)-aminopropyl)benzene

A 0.70 g portion of4-(3-Iodopropoxy)-1-(2-(N-t-butyloxycarbonyl)-aminopropyl)benzene,prepared above, was dissolved in 10 mL of anhydrous diethylether. Thisether solution was then added to a cold (4° C.) 250 mL saturatedammonia/ethanol solution.

The reaction was stirred and allowed to warm to room temperature. Themixture was resaturated with ammonia once a day for two days. Afterthree full days, the solvent was removed in vacuo and the residue wassuspended in 90 mL diethylether and 30 mL dichloromethane. The mixturewas washed with 5% potassium carbonate (pH 12), washed with brine, driedover magnesium sulfate, filtered, and evaporated to dryness in vacuo.The residue was purified by column chromatography over silica gel using84.5% dichloromethane, 15% methanol, and 0.5% acetic acid. Appropriatefractionswere pooled and solvents removed in vacuo to yield 0.44 g of athick, colorless oil.

4-[3-(5-Carboxyfluoresceinamidopropoxy)]-1-2-(N-t-butyl-oxycarbonyl)-aminopropyl)benzene

A 119 mg portion of 5-carboxy fluorescein and 38 mg portion ofN-hydroxysuccinimide was dissolved in 2 mL of anhydrousdimethylformamide.To this was added 68 mg ofN,N'-dicyclohexylcarbodiimide. After stirring for 3 hours, a mixture of100 mg4-(3-aminopropoxy)-1-(2-(N-t-butyloxy-carbonyl)-aminopropyl)benzene, 1mL of anhydrous dimethylformamide, and 100 microliters triethylaminewere added to the reaction mixture. After 18 hours of stirring, thesolvents were removed in vacuo to yield an orange solid. The product waspurified on four, 1.0 mm C18 reversed phase preparative thin layerchromatography plates developed in 69.5% methanol, 30.0% distilledwater, and 0.5% aceticacid. The band of R_(f) of 0.14 was collected andeluted with methanol toyield 115 mg of an orange solid.

4-[3-(5-Carboxyfluoresceinamidopopoxy]-1-(2-aminopropyl)benzene

The entire 115 mg yield of product from Example 22 was dissolved in 3 mLdichloromethane. With stirring 2 mL of trifluoroacetic acid was addeddropwise. After approximately 10 minutes, the solvents were removed invacuo, residue was dissolved in 5 mL methanol and 100 microliterstriethylamine and solvents removed in vacuo again. The residue wassuspended in 2 1/2 mL methanol and triethylamine was added until thesolidcompletely dissolved. The product was then purified on four 1.0 mmC18 reversed phase preparative thin layer chromatography platesdeveloped in 69.5% methanol, 30.0% distilled water, and 0.5% aceticacid. The band at R_(f) of 0.69 was collected and eluted with methanolto yield product.

EXAMPLE 4 Preparation of Methamphetamine Tracer Dimethylp-Phenylenediacetate

A 19.42 g portion of 1,4-phenylene diacetic acid was suspended in 200 mlofmethanol. To this was added 20 mL trimethyl orthoformate and 16 mL ofmethanol saturated with hydrogen chloride. After stirring for 3 hours atroom temperature, the solvents were removed in vacuo. The residue wasstirred with 1.5 l of hexane, mixed with 400 mL saturated sodiumbicarbonate solution and decanted into a separatory funnel leavingundissolved solids behind. The two phases were separated, the organicphase was washed with a second 400 ml saturated sodium bicarbonatesolution and then with 200 mL of brine. The resulting organic phase wasdried over magnesium sulfate, filtered, and evaporated to dryness invacuoto yield a white powder.

Methyl 4-Carboxymethylphenylacetate

Eight grams of dimethyl p-phenyleneacetate, prepared above, wasdissolved in 120 mL of methanol. To this was added 28.8 mL of 1M sodiumhydroxide solution and stirred at room temperature for 3 hours. Thereaction mixturewas then quenched by the addition of 8 mL acetic acidand evaporated to dryness in vacuo. The residue was diluted with 800 mLof saturated sodium bicarbonate solution and washed twice with 400 mLportions of hexane. The remaining aqueous phase was acidified to a pH of1 and extracted three times with 200 mL portions of dichloromethane. Thecombined organic extracts were washed with brine, dried over magnesiumsulfate, filtered, and evaporated to dryness in vacuo to yield 3.71 g ofa white solid.

4-(Carbomethoxymethyl)phenylacetyl chloride

A 3.04 g portion of methyl 4-carboxymethylphenylacetate, prepared above,was dissolved in 50 mL of anhydrous tetrahydrofuran. After the additionof2.5 mL oxalyl chloride, the mixture was stirred for 4 hours. Thesolvents were evaporated under a stream of dry nitrogen as the flask waswarmed to 45° C. Any remaining solvent or oxalyl chloride was thenremoved under high vacuum; the product was used without furtherpurification.

Methyl 4-(2-oxopropyl)-phenylacetate

A sample of copper (I) iodide was heated at 120° C. for 2 hours, thencooled to room temperature in a vacuum dessicator. Under a dry nitrogenatmosphere 50 mL anhydrous tetrahydrofuran was added to 8.35 g ofthecopper iodide. The suspension was chilled to -10° C., then 52 mLof 1.7Mmethyllithium in ether was quickly added dropwise. The entire yieldofthe 4(carbomethoxymethyl)phenylacetyl acid chloride was dissolved in 20mL anhydrous tetrahydrofuran and quickly added to the reaction mixtureas the temperature was lowered to -78° C. After 30 minutes the reactionwas quenched with 5 mL of methanol, warmed to room temperature, dilutedwith 15 mL methanol, and then diluted to 1 liter with diethylether. Thereaction was washed with saturated ammonium chloride andammoniumhydroxide, 10% ammonium chloride and ammonium hydroxide, and once withbrine. The organic phase was dried and evaporated to dryness in vacuo.The residue was purified by column chromatography over silica gel using30% ethylacetate and 70% hexane. Appropriate fractions were pooled andsolvents removed in vacuo to yield 0.60 g of product.

Methyl 4-(2-methylaminopropyl)phenylacetate

To 25 mL of methanol 0.59 g of methyl 4-(2-oxopropyl)phenylacetate,prepared above, was added and dissolved. Then with stirring thefollowing were added: 2.0 g methylamine hydrochloride, 0.3 g sodiumcyanoborohydride, and 0.3 mL of triethylamine. After approximately 12hours an additional 0.1 g of sodium cyanoborohydride was added to themixture. After 3 hours more, the reaction was diluted with 100 mL of0.1M hydrochloric acid, washed with diethylether, and the resultingaqueous phase was basified to a pH of 12 by addition of 5 mL portions of1N sodiumhydroxide solution. This was extracted three times with 100 mLportions of diethylether, combined extracts washed with brine and dried.The solvents were removed in vacuo to yield 0.44 g of a light yellowoil.

Methyl 4-(2-(N-t-butoxycarbonyl)-N-methylaminopropyl)phenyl-acetate

A 0.44 g portion of methyl, 4-(2-methylaminopropyl)phenylacetate,prepared above, was dissolved in 20 mL of methylene chloride. To thissolution was added 2.0 g di-t-butyldicarbonate and 0.45 mLtriethylamine. After 14 hours the reaction was diluted with 100 mL ofdiethylether and stirred with 40 mL of saturated sodium bicarbonatesolution. After 3 hours an additional 100 mL of diethylether was added,the organic phase was then separated and washed twice with 50 mLportions of brine. The resulting organic phase was dried and evaporatedin vacuo. The resulting yellow oil was purified by column chromatographyover silica gel using 35% ethylacetate and 65% hexane. The appropriatefractions were pooled and solvents evaporated in vacuo to yield 0.39 gof a viscous oil.

4-(2-(N-t-butoxycarbonyl)-N-methylaminopropyl)phenylacetic acid

A 320 mg portion of methyl4-(2-(N-t-butoxycarbonyl-N-methylamino-propyl))phenylacetate, preparedabove, was dissolved in 6 mL of dioxane. To this, 3 mL of 1N sodiumhydroxide solution was added. After 10 minutes of stirring, 3 mL ofaceticacid was added to the mixture, followed by 100 ml of diethylether.This waswashed once with 50 ml of brine and the brine was extracted withan equal volume of dichloromethane. The ether and dichloromethaneextracts were combined, dried over magnesium sulfate, filtered, andevaporated to dryness in vacuo to yield 360 mg of a light oil.

N-(Methylfluoresceinyl)-4-[2-(N-t-butyloxycarbonyl)methylaminopropyl]phenylacetamide

A 104 mg portion of 4-[2-(N-t-butoxycarbonyl)methylaminopropyl]phenylacetic acid, prepared above, and 94 mg of Woodward's Reagent K weredissolved in 3 mL of anhydrous dimethylformamide and 0.05 mLtriethylamine. After 1 hour of stirring at room temperature, 134 mg4'-aminomethylfluorescein dihydrochloride and 0.1 mL triethylamine wereadded. The mixture was stirred for 2 hours at room temperature and thenstored in a freezer overnight. The mixture was then warmed and stirredat room temperature for 3 hours before the solvent was removed in vacuo.The resulting orange solid was purified on four, 1.0 mm C18 reversedphase preparative thin layer chromatography plates developed in 74.5%methanol, 25% distilled water, and 0.5% acetic acid. The band at R_(f)0.21 was collected and eluted with methanol to yield product.

N-(Methylfluoresceinyl)-p-2-methylaminopropylphenylacetamide

The entire yield of the above-formed product was dissolved in 4 mL ofdichloromethane. While stirring two mL of trifluoroacetic acid wasstirredinto the solution, and after 5 minutes the solvent was removed invacuo. With stirring, the residue was dissolved in 25 mL methanol andtriethylamine was added dropwise until a dark orange color had formed.Thesolvent was removed again in vacuo and the residue dissolved inmethanol. The product was purified on four, 1.0 mm C18 reversed-phasepreparative thin layer chromatography plates developed in 64.5%methanol, 35.0% distilled water, and 0.5% acetic acid. The band at R_(f)0.50 was collected and eluted with methanol to yield product.

EXAMPLE 5 Amphetamine/Methamphetamine Assay A. Reagents

(1) Pretreatment Solution--A solution containing about 0.125 sodiumperiodate. (pH 4.5).

(2) Tracer: Consisting of Compound I prepared in Example 4 and CompoundII prepared in Example 3. Each compound is in 0.1M sodium phosphatebuffer atpH 7.5 containing 0.01% w/v bovine gamma globulin, and 0.1% w/vsodium azide.

(3) Antibody: Rabbitt or sheep antiserum consisting of antiserum raisedagainst amphetamine and methamphetamine appropriately diluted in 0.1Msodium phosphate buffer, 0.1% sodium azide and 2% ethylene glycol.

(4) Diluent buffer: 0.1M sodium phosphate, pH 7.5, 0.1% bovine gammaglobulin and 0.1% sodium azide.

(5) Calibrators: charcoal stripped human urine preserved wth 0.1% sodiumazide having d,l-amphetamine levels as follows: 0.00, 0.23, 0.38, 0.83,1.58, 3.08 ug/ml.

(6) Controls: charcoal stripped normal human urine preserved with 0.1%sodium azide, containing 0.68 or 2.08 ug/ml of d,l-amphetamine.

(7) Wash: A solution containing about 0.125M sodium periodate.

All polarized fluorescence measurements were made using the AbbottTDx®Clinical Analyzer.

B. Assay Protocol

(1) Equal portions of an unknown sample and pretreatment solution arepipetted into the predilute well. A sufficient volume of diluent bufferisadded to raise the volume to 500 ul. This mixture is incubated for 4-6minutes.

(2) A sample from the predilute well and 25 ul of antibody is pipettedintothe cuvette. A background intensity reading is taken.

(3) 25 ul each of tracer and antibody, and a sample from the predilutewell, is added to the cuvette. Sufficient diluent buffer is added toraisethe final volume to 2.0 mls.

(4) The fluorescence polarization due to tracer binding to the antibodyis obtained by subtracting the polarized fluorescence intensities of thebackground from the final polarized fluorescence intensities of themixture.

(5) The polarization values obtained are inversely proportional to theamphetamine and/or methamphetamine concentration of each sample.

(6) The polarization value for a sample is compared to a standard curveprepared using calibrators of known amphetamine or methamphetaminecontent.

EXAMPLE 6 Sodium Periodate Pretreatment

Samples containing 50 and 100 ug/ml of ephedrine were assayed with theAbbott TDx® Clinical Analyzer with and without the preincubationtreatment described in Example 5B above. The assay utilized theabove-described amphetamine/methamphetamine tracers and antibodies.Results are presented in Table II below:

                  TABLE II                                                        ______________________________________                                        μg/ml Ephedrine                                                                              Polarization                                                ______________________________________                                        50       w/pretreatment                                                                             216.09                                                  50       w/o pretreatment                                                                           79.75                                                   100      w/pretreatment                                                                             208.19                                                  100      w/o pretreatment                                                                           70.45                                                   ______________________________________                                    

The above results illustrate that sodium periodate treatment of sampleswithout the addition of pH raising constituents, such as base, iseffective in eliminating β-hydroxyphenethylamine cross-reactivity andisuseful for such purpose in amphetamine/methamphetamine fluorescencepolarization assays.

What is claimed is:
 1. In a method for determining phenethylamines in atest sample of biological fluid by fluorescence polarization assay theimprovement consisting of:pretreating said sample with an effectiveamount of an aqueous periodate solution having a pH from about 4.0 toabout 7.5 for a period of time sufficient to eliminateβ-hydroxyphenethylamine cross reactants.
 2. The improvement of claim 1wherein said sample is pretreated for about 1 to about 10 minutes. 3.The improvement of claim 1 wherein said aqueous periodate solutioncomprises from about 0.1 to about 0.25 Molar sodium periodate.
 4. Theimprovement of claim 1 wherein said sample is pretreated for about 4 toabout 5 minutes.
 5. The improvement of claim 1 wherein said pretreatmentis carried out at a temperature in a range from about 31° to about 36°C.
 6. A method for determining amphetamine and methamphetamine in a testsample of biological fluid by fluorescence polarization assay comprisingthe steps of:(a) pretreating said sample with an effective amount of anaqueous periodate solution having a pH from about 4.0 to 7.5 for aperiod of time sufficient to eliminate β-hydroxyphenethylamine crossreactants to amphetamine and methamphetamine; (b) intermixing saidpretreated sample with a salt of a first tracer of the formula: ##STR5##and a second tracer of the formula: ##STR6## wherein Q is a fluoresceinor a fluorescein derivative, T is SO₂, NH, HN(CH₂)₃ O, COCH₂, CO(CH₂)₂CONH, HN(CH₂)₂ or HN(CH₂)₂ NHCOCH₂, anda first antibody capable ofspecifically recognizing and binding amphetamine and said first tracerthereof; and a second antibody capable of specifically recognizing andbinding methamphetamine and said second tracer thereof; and (c) thendetermining the amount of each tracer bound to said first and secondantibodies by fluorescence polarization techniques as a measure of theamount of amphetamine and methamphetamine in the sample.
 7. The methodof claim 6, wherein said fluorescein derivative of said first tracer isa carboxyfluorescein and said fluorescein derivative of said secondtracer is 4'-aminomethylfluorescein.
 8. The method of claim 6, whereinsaid first tracer has a formula: ##STR7##
 9. The method of claim 6,wherein said second tracer has a formula: ##STR8##
 10. A stabilizedreagent kit useful in determination of amphetamine and methamphetaminecomprising:(a) a pretreatment solution having an amount of aqueoussodium periodate effective in eliminating β-hydroxyphenethylaminecrossreactivity to amphetamine and methamphetamine; (b) a fluorescencereagent having salts of a first and a second tracer, said first tracerbeing a fluorescein or fluorescein derivative compound bound to a ligandanalog of amphetamine and said second tracer being a fluorescein orfluorescein derivative compound bound to a ligand analog ofmethamphetamine; and (c) an antibody reagent having a first antibodycapable of specifically recognizing amphetamine and said first tracerthereof and a second antibody capable of specifically recognizingmethamphetamine and said second tracer thereof.
 11. The stabilizedreagent kit of claim 10, further comprising an aqueous wash solutionhaving from about 0.1 to about 0.25 molar sodium periodate.
 12. Thestabilized reagent kit of claim 10, wherein said first tracer has aformula ##STR9## and said second tracer has a formula ##STR10## whereinQ is said fluorescein or fluorescein derivative; T is SO₂, HN, HN(CH₂)₃O, COCH₂, CO(CH₂)₂ CONH, HN(CH₂)₂ or HN(CH₂)₂ NHCO CH₂.
 13. Thestabilized reagent kit of claim 10, wherein said first tracer has aformula ##STR11## and said second tracer has a formula ##STR12##
 14. Thestabilized reagent kit of claim 10, wherein said first antibody israised in response to an immunogen having the structure: ##STR13## andsaid second antibody is raised in response to an immunogen having thestructure: ##STR14## wherein Q is a poly(amino acid) or poly(amino acid)derivative.
 15. A method for determining amphetamine and methamphetaminein test samples of biological fluid by fluorescence polarization assayutilizing an automatic assay apparatus having a sample and reagentdispensing means comprising the steps of:(a) pretreating a sample toeliminate β-hydroxyphenethylamine cross-reactivity using only aneffective amount of an aqueous periodate solution having a pH from about4.0 to 7.5 for a period of time sufficient to eliminate saidβ-hydroxyphenethylamine cross-reactivity; (b) intermixing saidpretreated sample with a salt of a first tracer of the formula:##STR15## and a second tracer of the formula: ##STR16## wherein Q isfluorescein or a fluorescein derivative, T is SO₂, NH, HN(CH₂)₃ O,COCH₂, CO(CH₂)₂ CONH, HN(CH₂)₂ or HN(CH₂)₂ NHCOCH₂, anda first antibodycapable of specifically recognizing and binding amphetamine and saidfirst tracer thereof; and a second antibody capable of specificallyrecognizing and binding methamphetamine and said second tracer thereof;(c) determining the amount of each tracer bound to said first and secondantibodies by fluorescence polarization techniques as a measure of theamount of amphetamine and methamphetamine in the sample; and (d) washingsaid dispensing means in a 0.1 to 0.25M aqueous periodate solution tominimize sample adhesion to said dispensing means.
 16. The method ofclaim 15, wherein said dispensing means is a probe which is washed insaid periodate solution.